(PQC2) Niche-responsive RNA editing by ADAR1 in dormant multiple myeloma initiating cell maintenance



 DESCRIPTION (provided by applicant): Despite new available therapies, including immunomodulatory drugs such as thalidomide and lenalidomide in multiple myeloma (MM), about 20-25% of patients are still considered at high risk for treatment failure. The emergence of therapeutically recalcitrant cases and disease relapse even under intensive treatment regimens suggests the existence of a dormant myeloma-initiating population within the bone marrow (BM) that is capable of drug escape. These myeloma-initiating cells are defined by the lack of the plasma cell marker CD138, while they express several hematopoietic stem (CD38) and B cell markers such as CD27 and CD19; however, the molecular and cellular mechanisms that regulate myeloma-initiating cell generation and maintenance are so far poorly understood. Recently, we and other groups showed that both NOTCH signaling and inflammation- responsive ADAR1 activation are crucial events regulating malignant stem cell maintenance in the bone marrow microenvironment, characterized by enhanced survival and self-renewal and cell cycle alterations of dormant progenitor cells. In this context, the central hypothesis of this proposal is that MM niche-derived pro- inflammatory signals induce aberrant human-specific RNA editing driven by adenosine deaminase acting on dsRNA-1 (ADAR1) in dormant myeloma-initiating cells that is accentuated by lenalidomide resistance. This project will: 1) determine whether ADAR1 activity is enhanced in myeloma-initiating cells and investigate the effects of lenalidomide treatment on ADAR1-dependent RNA editing in therapeutic resistance and relapse; 2) identify the NOTCH-regulated pro-inflammatory cytokines that activate ADAR1-dependent RNA editing in myeloma-initiating cells; and 3) determine whether direct inhibition of ADAR1 activity in MM initiating cells, or blocking microenvironmental signals that activate ADAR1, sensitizes myeloma-initiating cells to lenalidomide and prevents myeloma-initiating cell maintenance. These aims will address PQC2: What molecular or cellular events establish tumor dormancy after treatment and what leads to recurrence? We will utilize both in vitro and in vivo measures of myeloma-initiating cell function using multicolor flow cytometry and fluorescent RNA probe-based strategies to purify and profile primary MM cellular constituents, coupled with a novel diagnostic qPCR-based assay to detect endogenous RNA editing, and gene knockout (CRISPR) or lentiviral shRNA- knockdown strategies to modulate NOTCH-dependent ADAR1 activation. A fluorescent ubiquitination cell cycle indicator (FUCCI) bi-cistronic lentiviral reporer will facilitate investigations of dormant live cells, in robust bone marrow stromal co-culture models and bioluminescent humanized MM mouse models. The ultimate goal is to investigate niche-dependent ADAR1 activation as a novel mechanism driving transcriptome recoding and molecular evolution of dormant myeloma-initiating cells, laying the groundwork for targeted therapeutics with potential applications in an array of other therapeutically recalcitrant malignancies.